Bank of stem cells for producing cells for transplantation having hla antigens matching those of transplant recipients and methods for making and using such a stem cell bank

ABSTRACT

Methods for producing stem cell banks, preferably human, which optionally may be transgenic, e.g., comprised of homozygous MHC allele cell lines are provided. These cells are produced preferably from parthenogenic, IVF, or same-species or cross-species nuclear transfer embryos or by de-differentiation of somatic cells by cytoplasm transfer. Methods for using these stem cell banks for producing stem and differentiated cells for therapy, especially acute therapies, and for screening for drugs for disease treatment are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/721,618 filed Dec. 20, 2012, now issued as U.S. Pat. No.10,047,340; which is a continuation application of U.S. application Ser.No. 10/445,195 filed May 27, 2003, now abandoned; which claims thebenefit under 35 USC § 119(e) to U.S. Application Ser. No. 60/448,585filed Feb. 21, 2003 and to U.S. Application Ser. No. 60/382,616 filedMay 24, 2002, both now expired. The disclosure of each of the priorapplications is considered part of and is incorporated by reference inthe disclosure of this application.

BACKGROUND OF THE INVENTION Field of the Invention

The invention described herein relates to methods for producing acollection of human and non-human stem cell cultures, preferably humanstem cell cultures, each of which contains totipotent or pluripotentstem cells that have genes encoding the same set of critical cellsurface antigenic proteins, e.g., histocompatibility antigens (e.g., HLAantigens in the case of human) as are present on the cells of members ofa human population. (By critical antigens is meant the set of antigensthat form the major histocompatibility complex and other antigens suchas blood group antigens that are involved in immuno-mediated rejectionwhen collogenic cells and tissues are transplanted into donors thatexpress a different set of histocompatibility and other criticalantigens). The methods disclosed herein include deriving such human stemcell cultures from cells of early embryos produced e.g., by in vitrofertilization, parthenogenesis, and by nuclear transfer. Also, stemcells can be produced by transfer of cytoplasm from embryonic cells,e.g., oocytes, early embryonic cells or ES cells into somatic cells.

The invention described herein also relates to methods wherein suchhuman and non-human stem cell cultures are induced to differentiate exor in vivo into cell types that are useful for therapeutic celltransplantation; and to methods by which the differentiated cells areisolated from other cell types. The invention also relates to methods inwhich stem cell-derived differentiated cells having a selected set ofcritical cell surface antigens are therapeutically transplanted orengrafted to a recipient, e.g., a human patient in need of a celltransplant having cells that express the same critical cell surfaceantigens. The invention further relates to a collection or “bank” ofcultures of different types of stem cells, each culture having adifferent set of genes encoding cell surface antigenic proteins presentin a human population; to compositions comprising the individual stemcell cultures that make up such a stem cell bank; and to compositionscomprising differentiated cells derived from such stem cells.

Preferably, stem cell banks produced according to the invention willcomprise stem cell lines which are homozygous for MHC alleles whichoccur very frequently in the human population. Typically, a stem cellbank according to the invention will comprise at least 15 stem celllines and more preferably at least 100 to 1000 stem cell lines. Thereby,the stem cell bank will provide maximal therapeutic and diagnosticefficacy as it will contain cells that are histocompatible for a widerange of potential transplant recipients.

Background Information A. Histocompatibility and Transplant Rejection

Histocompatibility is a largely unsolved problem in transplant medicine.Rejection of transplanted tissue is the result of an adaptive immuneresponse to alloantigens on the grafted tissue by the transplantrecipient. The alloantigens are “non-self proteins, i.e., antigenicproteins that vary among individuals in the population and areidentified as foreign by the immune system of a transplant recipient.The antigens on the surfaces of transplanted tissue that most stronglyevoke rejection are the blood group (ABO) antigens and the majorhistocompatibity complex (MHC) proteins and in the case of humans, thehuman leukocyte antigen (HLA) proteins.

The blood group antigens were first described by Landsteiner in 1900;they are branched oligosaccharides that are attached to proteins andlipids on the surfaces of red blood cells, endothelial cells, and othercells, and are also present in secretions such as saliva. Compatibilityof the blood group antigens of the ABO system of a vascularized organ ortissue transplant with those of the transplant recipient is generallyrequired; but blood group compatibility may be unnecessary for manytypes of cell transplants.

The HLA proteins are encoded by clusters of genes that form a regionlocated on chromosome 6 known as the Major Histocompatibility Complex,or MHC, in recognition of the important role of the proteins encoded bythe MHC loci in graft rejection. Accordingly, the HLA proteins are alsoreferred to as MHC proteins. The MHC genes and proteins will be usedinterchangeably in this application as the application encompasses humanand non-human animal applications. The HLA or MHC proteins normally playa role in defending the body against foreign pathogens such as viruses,bacteria, and toxins. They are cell surface glycoproteins that bindpeptides at intracellular locations and deliver them to the cellsurface, where the combined ligand is recognized by a T cell. Class IMHC proteins are found on virtually all of the nucleated cells of thebody. The class I MHC proteins bind peptides present in the cytosol andform peptide-MHC protein complexes that are presented at the cellsurface, where they are recognized by cytotoxic CD8+ T cells. Class IIMHC proteins are usually found only on antigen-presenting cells such asB lymphocytes, macrophages, and dendritic cells. The class II MHCproteins bind peptides present in a cell's vesicular system and formpeptide-MHC protein complexes that are presented at the cell surface,where they are recognized by CD4+ T cells. CD4+ T cells activated byclass II MHC proteins undergo clonal expansion with production ofregulatory cytokines that signal helper and cytotoxic T cells.Unfortunately for those in need of transplants, the frequency of T cellsin the body that are specific for non-self MHC molecules is relativelyhigh, with the result that differences at MHC loci are the most potentcritical elicitors of rejection of initial grafts. Rejection of mosttransplanted tissues is triggered predominantly by the recognition ofclass I MHC proteins as non-self proteins. T cell recognition of foreignantigens on the transplanted tissue sets in motion a chain of signalingand regulatory events that causes the activation and recruitment ofadditional T cells and other cytotoxic cells, and culminates in thedestruction of the transplanted tissue. (Charles A. Janeway et al.,Immunobiology. Garland Publishing, New York, N.Y., 2001, p. 524).

B. The Genes Encoding MHC Proteins

The MHC genes are polygenic—each individual possesses multiple,different MHC class I and MHC class II genes. The MHC genes are alsopolymorphic—many variants of each gene are present in the human andnon-human population. In fact, the MHC genes are the most polymorphicgenes known. Each MHC Class I receptor consists of a variable a chainand a relatively conserved β2-microglobulin chain. Three different,highly polymorphic class I a chain genes have been identified. These arecalled HLA-A, HLA-B, and HLA-C. Variations in the a chain chains accountfor all of the different class I MHC genes in the population. MHC ClassII receptors are also made up of two polypeptide chains, an a chain anda β chain, both of which are polymorphic. In humans, there are threepairs of MHC class II a and β chain genes, called HLA-DR, HLA-DP, andHLA-DQ. Frequently, the HLA-DR cluster contains an extra gene encoding aβ chain that can combine with the DR a chain; thus, an individual'sthree MHC Class II genes can give rise to four different MHC Class IImolecules.

In humans, the genes encoding the MHC class I a chains and the MHC classII a and β chain are clustered on the short arm of chromosome 6 in aregion that extends over from 4 to 7 million base pairs that is calledthe major histocompatibility complex. Every person usually inherits acopy of each HLA gene from each parent. If an individual's two allelesfor a particular MHC locus encode structurally different proteins, theindividual is heterozygous for that MHC allele. If an individual has twoMHC alleles that encode the same MHC molecule, the individual ishomozygous for that MHC allele. Because there are so many differentvariants of the MHC alleles in the population, most people haveheterozygous MHC alleles. The numbers of different alleles found foreach type of MHC class I a chain and MHC class II α and β chains as ofJanuary 2003 are shown in Table 1.

TABLE 1 The numbers of different alleles for the polymorphic MHC class Iand class II chains identified as of January, 2003. MHC Chain No. ofAlleles HLA-A 266 HLA-B 511 HLA-C 6 HLA-DRA 3 HLA-DRB 403 HLA-DQA1 23HLA-DQB1 53 HLA-DPA1 20 HLA-DPB1 101

The data in Table 1 is from the Internet web site of the InformaticsGroup of the Anthony Nolan Trust, The Royal Free Hospital, Hampstead,London, England. Lists of identified HLA Class I and Class II allelesare also available at the same web site.

C. Matching MHC Types to Inhibit Rejection of Transplants

Since the recognition that-non-self-M1-G-molecules are a majordeterminant of graft rejection, much effort has been put into developingassays to identify the MHC types present on the cells of tissue to betransplanted, and on the cells of transplant recipients, in order tomatch the types of MHC molecules present in the transplant tissue withthose of the recipient. Tissue typing, the detection of MHC antigens, isperformed by various means; for example, (i) by serology, usingantibodies specific for particular MHC molecules to detect the presenceof the targeted MHC molecules on donor or recipient cells, e.g., by thelymphocytotoxicity test; (ii) by detection of antibodies of a transplantrecipient that bind specifically to a MHC protein of transplant tissue;and (iii) by direct analysis of the nucleotide sequence of the DNA ofthe MHC alleles. Most tissue typing for organ banking purposes is doneby determining the blood type (ABO typing) and by typing the patient'sand donor cells using serological methods; however, the use of rapid andreliable DNA-specific methods is increasing. Such methods can employsequence-specific oligonucleotide primers and amplification by thepolymerase chain reaction (PCR), and can be augmented by combiningfluorescent detection methods with the use of a DNA chip to which arebound sequence specific oligonucleotides designed to detect uniquesequences present in the different MHC alleles.

At present, tissue typing to match the HLA antigens of a transplant withthose of a recipient is usually limited to the Class I HLA-A and -Bantigens, and the Class II HLA-DR antigens. Most transplant donors areunrelated to the transplant recipient, and finding a tissue type tomatch that of the recipient usually involves matching the blood type andas many as possible of the 6 HLA alleles—two for each HLA-A, —B, and -DRlocus. Transplant centers do not usually consider potentialincompatibilities at other FILA loci, such as HLA-C and HLA-DPB1,although mismatches at these loci can also contribute to rejection.Considering only the combinations of maternal and paternal alleles ofthe HLA-A. HLA-B, and HLA-DR loci identified to date, there is acomplexity of billions of possible tissue types. The task of matchingHLA types of organs for transplant is simplified in that HLA-A and HLA-Bare usually identified serologically. The number of HLA antigensidentified serologically is considerably less than the number ofdifferent MLA antigens based on DNA sequencing. The World HealthOrganization (WHO) has recognized 28 distinct antigens in the HLA-Alocus and 59 in the HLA-B locus, based on serological typing. Matchingorgans is also simplified to some extent by the fact that some allelesare much more common than others. Some of the more common HLA-A andHLA-B alleles are shown in Table 2:

TABLE 2 Frequency of Common HLA-A and HLA-B Alleles in the PopulationHLA-A (Frequency (%)) HLA-B (Frequency (%)) HLA-A 1 (25.1) HLAB5 (15.2)HLA-A2 (44.8) HLA-B 7 (18.2) HLA-A3 (22.6) HLA-B 8 (16.7) HLA-A24 (18.2)HLA-B 12 (32.5) HLA-A 11 (11.8) HLAB14 (8.8) HLA-A28 (9.8) HLA-B 18(11.3) HLA-A29 (10.3) HLA-B35 (15.2) HLA-A3 2 (9.8) HLA-B40 (13.7) HLA-B15 (12.3) (from Snell G D et al, Histocompatibility, New York, AcademicPress, 1976)

The frequencies with which the various alleles appear in a population isnot random; it depends on the racial makeup of the population. Dr.Motomi Mori has determined the frequencies with which thousand ofdifferent haplotypes of HLA-A, —B, and -DR loci appear in Caucasian,African-American, Asian-American, and Native American populations. Eachhaplotype is a particular combination of HLA-A, HLA-B, and HLA-DR locithat is present on a single copy of chromosome no. 6. The frequencies ofseveral relatively common HLA-A, —B, and -DR haplotypes are shown inTable 3 to illustrate the wide variation in HLA haplotype frequencies insome of the racial groups that make up the North American population. Ininterpreting haplotype frequency data such as that shown in Table 3, onemust bear in mind that cells of patients and organs are diploid and havean HLA type that is the product of the HLA haplotypes of the chromosomesinherited from both parents.

TABLE 3 Examples of HLA-A, -B, -DR haplotype frequencies HLA-A, -B, and-DR haplotype frequencies (expressed in percent) and their respectiverankings within each racial group: Caucasian (CAU), African-American(AFR), Asian-American (ASI) and Native American (NAT). HaplotypeFrequency (%) Ranking A B DR CAU afr ASI LAT NAT CAU AFR ASI LAT NAT 1 72 0.5349 0.2094 0.0798 0.1888 0.2812 21 58 262 91 62 1 8 3 5.1812 1.24910.3195 1.6733 4.7439 1 2 54 3 1 2 14 1 0.1563 0.0444 0.0076 0.37940.0624 107 539 1451 39 312 2 35 4 0.1457 0.0737 0.3293 1.2858 0.6342 115302 49 4 12 2 35 8 0.0823 0.0931 0.1756 1.7641 0.3289 241 226 122 1 46 244 4 2.1507 0.6506 0.1276 0.6906 2.0004 3 4 170 12 3 3 7 2 2.6285 0.75960.1891 1.1986 2.7083 2 3 113 5 2 3 7 4 0.4411 0.1534 0.0498 0.17950.4448 30 104 408 98 29 3 7 8 0.0848 0.0367 0.0000 0.0622 0.0537 230 65314053 310 366 3 35 1 1.0224 0.2741 0.1372 0.3552 0.8125 7 29 156 44 8 3151 4 0.0915 0.0342 0.1646 0.2597 0.5691 209 699 135 64 16 32 14 7 0.26170.0513 0.0046 0.1324 0.1775 57 479 1858 140 104

The data in Table 3 was produced for The National Marrow Donor ProgramDonor Registry, and is available at the Internet web site of MotomiMori, Ph.D., Huntsman Cancer Institute, Salt Lake City, Utah.

D. Rejection Triggered by Minor Histocompatibility Antigens

Matching the MHC molecules of a transplant to those of the recipientsignificantly improves the success rate of clinical transplantation;however, it does not prevent rejection, even when the transplant isbetween HLA-identical siblings. This is because rejection is alsotriggered by differences between the minor histocompatibility antigens.These polymorphic antigens are actually “non-self peptides bound to MHCmolecules on the cells of the transplant tissue. The rejection responseevoked by a single minor histocompatibility antigen is much weaker thanthat evoked by differences in MHC antigens, because the frequency of theresponding T cells is much lower (Janeway et al., supra, page 525).Nonetheless, differences between minor histocompatibility antigens willoften cause the immune system of a transplant recipient to eventuallyreject a transplant, even where there is a match between the MHCantigens, unless immunosuppressive drugs are used.

E. Inadequate Supply of Cells, Tissues, and Organs for Transplant

The number of people in need of cell, tissue, and organ transplants isfar greater than the available supply of cells, tissues, and organssuitable for transplantation. Under these circumstances, it is notsurprising that obtaining a good match between the MHC proteins of arecipient and those of the transplant is frequently impossible, and manytransplant recipients must wait for an MHC-matched transplant to becomeavailable, or accept a transplant that is not MHC-matched. If the latteris necessary, the transplant recipient must rely on heavier doses ofimmunosuppressive drugs and face a greater risk of rejection than wouldbe the case if MHC matching had been possible. There is presently agreat need for new sources of cells, tissues, and organs suitable fortransplantation that are histocompatible with the patients in need ofsuch transplants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows cynomolgus monkey blastocysts derived from parthenogeneticembryos.

FIG. 2 shows an ES-like cell line (Cyno 1) derived from a cynomolgusparthenogenetic blastocyst.

FIG. 3 shows the Cyno 1 cell line before and after immunosurgery.

FIG. 4 shows the Cyno 1 cell line before and after immunosurgery.

FIG. 5 shows the Cyno 1 cell line 5 days after plating.

FIG. 6 shows the Cyno 1 cell line growing on top of a feeder layer.

FIG. 7 shows the results of an RT-PCR showing that the Cyno-1 cell lineis homozygous for the Snrpn gene (contains paternal allele).

FIG. 8 shows metaphase II oocytes at retrieval.

FIG. 9 shows 4 and 6 cell embryo 48 hours after parthenogeneticactivation.

FIG. 10 shows blastocoele cavities in human parthenogenetic activation48 hours after activation.

FIG. 11 shows human parthenogenetic embryo having an inner cell mass.

FIG. 12 shows human ES-like cells derived from cultured ICM cells.

DETAILED DESCRIPTION OF THE INVENTION A. A Bank of Stem Cell LinesHomozygous for MHC Loci

It is an object of the present invention to prepare a bank oftotipotent, nearly totipotent, and/or pluripotent stem cell lines thatare homozygous for one or more critical antigen genes, i.e., genes whichencode histocompatibility antigens, e.g., in the case of human stemcells and “stem-like” cells, MHC alleles that are present in the humanpopulation. Preferably, this work will be homozygous for MHC allelesthat are representative of at least most prevalent in the particularspecies, preferably human. Many of these lines will also have an ABOblood group type 0-negative to make them broadly compatible across thedifferent blood types. Stem cell lines of the present invention can beinduced to differentiate into cell types suitable for therapeutictransplant. Because the cells of the present invention have homozygousMHC alleles, the chance of obtaining cells for transplant that have MHCalleles that match those of a patient in need of a transplant issignificantly enhanced. Instead of having to find a six of six matchbetween two sets of HLA-A, HLA-B, and HLA-DR antigens, a high level ofhistocompatibility is provided by the cells for transplant of thepresent invention when either of the two HLA-A, HLA-B, and HLA-DRantigens of the prospective transplant recipient matches one of thecorresponding homozygous HLA antigens of the cells for transplant. Forexample, a stem cell bank able to provide cells having an HLA-A/HLA-Bmatch to a patient having any of the eight HLA-A and nine HLA-B antigenslisted in Table 2 would require only 72 stem cell lines with homozygousHLA-A and HLA-B antigens; whereas a bank of stem cells with heterozygousHLA-A and HLA-B antigens would need to have 4032 different stem celllines. To provide a library of heterozygous stem cell lines that matchthe WHO list of serological types would require obtaining stem cellshaving every combination of 28 different pairs of HLA-A antigens and 59different pairs of HLA-B, to account for both the maternal and paternalalleles for each loci. The complexity of such a stem cell bank, i.e.,the number of different cell lines required, would be 2,587,032. Incontrast, a bank of stem cells homozygous for the same HLA-A and HLA-Bantigens would only need to have a complexity of 1,652 stem cell linesto guarantee a match to a patient with HLA-A and -B antigens on the WHOlist of serological types. The actual number required to meet the needsof a majority of patients will actually be less than this due to thenonrandom distribution of alleles in various populations around theworld. Patients in need of bone marrow stem cell grafts who arehomozygous in particular alleles are particularly sensitive to graftversus host disease when heterozygous bone marrow grafts are used. Stemcell grafts using stem cells having homozygous alleles made according tothe methods of the present invention would alleviate this commoncomplication of transplants.

This present invention provides novel means for making libraries oftotipotent and/or pluripotent stem cells that can serve as sources ofcells for therapeutic transplant that are highly histocompatible withhuman or nonhuman patients in need of cell transplants. Additionally,those cell lines are useful in creating animal models for specificdiseases that may be used to evaluate potential treatments and drugantidotes. In one embodiment, the invention comprises preparing a bankof stem cell lines that are homozygous for one or more critical antigenalleles, in the case of human stem cells. MHC alleles that are presentin all or most of the world's populations, including the populations ofNorth America, Central and South America, Europe, Africa, and Asia, andthe Pacific islands. It is an object of the present invention to providea stem cell bank comprising stem cells generated from vertebrate somaticcells, preferably mammalian somatic cells, and more preferably humanresearch cells that are homozygous for one or more critical antigenalleles, e.g., MHC alleles using nuclear transfer or parthenogenicproduced embryos. A preferred object of the present invention is toprovide a stem cell bank comprising diploid vertebrate, preferablymammalian and more preferably human stem cells generated byparthenogenesis that are homozygous for MHC alleles. Another object ofthe present invention is to provide a stem cell bank comprising diploidvertebrate, preferably mammalian and more preferably human stem cellsgenerated by union of sperm and egg in vitro that are homozygous for oneor more MHC alleles. S&M further, an object of the invention is topreview a bank of homozygous IES cell lines by introducing cytoplasmfrom embryonic cells into growth cells that are homozygous for specificMITC allele or are rendered homozygous by genetic manipulation. (Theembryonic cytoplasm contains constituents that de-differentiate thedifferentiated growth cell into stem cell lineages.

The stem cell bank of the present invention comprises lines oftotipotent, nearly totipotent, and/or pluripotent stem cells that arehomozygous for at least one histocompatibility antigen collection. Inthe case of human stem cells this will be an MHC allele selected fromthe group consisting of HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, and HLA-DP.In a useful embodiment, the stem cell bank comprises totipotent, nearlytotipotent, and/or pluripotent stem cells stem cells that are homozygousfor the significant histocompatibility antigen alleles, e.g., the HLA-A,HLA-B, and HLA-DR alleles. In another embodiment, the stem cell bankcomprises stem cells that are homozygous for all of thehistocompatibility antigen alleles, e.g., MHC alleles.

The stem cell bank of the present invention comprises totipotent and/ornearly totipotent stem cells such as embryonic stem (ES) cells, that candifferentiate in vivo or ex vivo into a wide variety of different celltypes having one or more homozygous MHC alleles. The stem cell bank ofthe present invention can also comprise partially differentiated,pluripotent stem cells such as neuronal stem cells and/or hematopoieticstem cells, that differentiate in vivo or ex vivo into a more limitednumber of differentiated cell types having one or more homozygous MHCalleles. These stem cells optionally may be transgenic, e.g., they mayexpress antigens that encode therapeutic or diagnostic proteins andpolypeptides. For example, the stem cells may be genetically engineeredto express proteins that inhibit immune rejection responses such asCD4O-L (CD154 or gp139) or in the case of porcine stem cells May begenetically engineered to knock-out a glycosylated antigen that is knownto trigger immune rejection responses.

An object of the present invention is to provide a stem cell bankcomprising stem cells having homozygous histocompatibility alleles,i.e., MHC alleles that are available “off the shelf” for providinghistocompatible cells suitable for transplant to patients in need ofsuch a transplant. Desirably, this stem bank will include stem celllines that are representative of the different histocompatibilityantigens expressed in the particular species, e.g., human. In a usefulembodiment, the stem cell bank comprises stem cells that are isolatedand maintained without feeder cells or serum of non-human animals, tominimize concerns of contamination by pathogens. In another usefulembodiment, the stem cell bank comprises stem cells that are geneticallymodified relative to the cells of the donor, e.g., human donor fromwhich they are derived. In another embodiment, the stem cell bankcomprises stem cells generated by nuclear transfer techniques that arerejuvenated, or “hyper-youthful,” relative to the cells of the donor,e.g., non-human mammal or human donor from which they are derived, andalso relative to age-matched control cells of the same type and speciesthat are not generated by nuclear transfer techniques. Such rejuvenatedor “hyper-youthful” cells have extended telomeres, increasedproliferative life-span, and metabolism that is more characteristic ofyouthful cells, e.g., increased EPC-1 and telomerase activities,relative to the human donor cells from which they are derived, and alsorelative to age-matched control cells of the same type that are notgenerated by nuclear transfer techniques.

Another object of the present invention is to provide a stem cell bankcomprising stem cells having homozygous recessive alleles responsiblefor genetically inherited diseases. Recessive disease-causing genes areendemic in the population, and such stem cells can be generated byparthenogenesis using oocytes collected from female carriers of therecessive disease-causing alleles. There is great need for totipotent,nearly totipotent, and/or pluripotent stem cells that having homozygousrecessive disease-causing alleles that can be induced to differentiateinto cells useful for basic research directed to studying the diseasephenotype, both ex vivo and in vivo (e.g., in immunodeficient laboratoryanimals), and for screening to discover drugs and other therapies thattreat or cure the disease.

B. Terms Used in Describing the Invention

As used herein, a “stem cell” is a cell that has the ability toproliferate in culture, producing some daughter cells that remainrelatively undifferentiated, and other daughter cells that give rise tocells of one or more specialized cell types; and “differentiation”refers to a progressive, transforming process whereby a cell acquiresthe biochemical and morphological properties necessary to perform itsspecialized functions. Stem cells therefore reside immediatelyantecedent to the branch points of the developmental tree.

As used herein, a “totipotent” cell is a stem cell with the “total powerto differentiate into any cell type in the body, including the germ linefollowing exposure to stimuli like that normally occurring indevelopment. Examples of totipotent cells include an embryonic stem (ES)cell, an embryonic germ (EG) cell, an inner cell mass (ICM)-derivedcell, or a cultured cell from the epiblast of a late-stage blastocyst.

As used herein, a “nearly totipotent cell” is a stem cell with the powerto differentiate into most or nearly all of the cell types in the bodyfollowing exposure to stimuli like that normally occurring indevelopment.

As used herein, a “pluripotent cell” is a stem cell that is capable ofdifferentiating into multiple somatic cell types, but not into most orall cell types. This would include by way of example, but not limitedto, mesenchymal stem cells that can differentiate into bone, cartilageand muscle; hematopoietic stem cells that can differentiate into blood,endothelium, and myocardium; neuronal stem cells that can differentiateinto neurons and glia; and so on.

As used herein, an “embryonic stem cell line is a cell line” with thecharacteristics of the murine ES cells isolated from morulae orblastocyst inner cell masses, as reported by Martin (Proc. Natl. Acad.Sci. USA (1981) 78:7634-7638); and by Evans et al. (Nature (1981) 292:154-156). ES cells have high nuclear-to-cytoplasm ratio, prominentnucleoli, are capable of proliferating indefinitely and can bedifferentiate into most or all of the specialized cell types of anorganism, such as the three embryonic germ layers, all somatic celllineages, and the germ line. ES cells that can differentiate into all ofthe specialized cell types of an organism are totipotent. In some cases,ES cells are obtained that can differentiate into almost all of thespecialized cell types of an organism; but not into one or a smallnumber of specific cell types. For example, Thomson et al. describeisolating a primate ES cell that, when transferred into anotherblastocyst, does not contribute to the germ line (Proc. Natl. Acad. Sci.USA. (1995) 92:7844-7848). Such ES cells are an example of stem cellsthat are nearly totipotent.

As used herein, “inner cell mass-derived cells” (ICM-derived cells) arecells directly derived from isolated ICMs or morulae without passagingthem to establish a continuous ES or ES-like cell line. Methods formaking and using ICM-derived cells are described in co-owned U.S. Pat.No. 6,235,970, the contents of which are incorporated herein in theirentirety.

As used herein, “enucleation” refers to removal of the genomic DNA froman cell, e.g., from a recipient oocyte. Enucleation therefore includesremoval of genomic DNA that is not surrounded by a nuclear membrane,e.g., removal of chromosomes aligned to form a metaphase plate. Asdiscussed below, the recipient cell can be enucleated by any of theknown means either before, concomitant with, or after nuclear transfer.

As used herein, “ex vivo” cell culture refers to culturing cells outsideof the body. Ex vivo cell culture includes cell culture in vitro, e.g.,in suspension, or in single- or multi-well plates. Ex vivo culture alsoincludes co-culturing cells with two or more different cell types, andculturing in or on 2- or 3-dimensional supports or matrices, includingmethods for culturing cells alone or with other cell types to formartificial tissues.

As used herein, “parthenogenetic embryos” refers to an embryo that onlycontains male or female chromosomal DNA that is derived from male orfemale gametes. For example, parthenogenetic embryos can be derived byactivation of unfertilized female gametes, e.g., unfertilized human,murine, cynomolgus or rabbit oocytes.

As used herein, “nuclear transfer embryo” refers to an embryo that isproduced by the fusion or transplantation of a donor cell or DNA from adonor cell into a suitable recipient cell, typically an oocyte of thesame or different species that is treated before, concomitant or aftertransplant or fusion to remove or inactivate its endogenous nuclear DNA.The donor cell used for nuclear transfer include embryonic anddifferentiated cells, e.g., somatic and germ cells. The donor cell maybe in a proliferative cell cycle (G₁, G₂, S or M) or non-proliferating(G₀ or quiescent). Preferably, the donor cell or DNA from the donor cellis derived from a proliferating mammalian cell culture, e.g., afibroblast cell culture. The donor cell optionally may be transgenic,i.e., it may comprise one or more genetic addition, substitution ordeletion modifications.

As used herein, the term “gene” refers to the nucleotide sequences at agenetic locus that encode and regulate expression of a functional mRNAmolecule or a polypeptide; i.e., as used herein, a gene includes thenucleotide sequences that make up the coding sequence (exons andintrons), the promoter, enhancers, and other DNA elements that regulatetranscription, including as elements conferring cell type-specific anddifferentiation stage-specific expression, hormone responsive elements,repressor elements, etc., and nucleotide sequences that encode signalsthat regulate splicing and translation of the mRNA, such as a cleavagesignal, a polyadenylation signal, or an internal ribosome entry site(IRES).

C. Providing Histocompatible Transplants to Animal or Human Recipients

Another object of the invention is to provide a method by which a humanor non-human animal, e.g., a person in need of a cell or tissuetransplant can be provided with cells or tissue suitable fortransplantation that have homozygous histocompatibility antigen alleles,e.g., in the case of human recipients MHC alleles that match the MHCalleles of the person needing the transplant. The invention provides amethod in which the MHC alleles of a person in need of a transplant (therecipient) are identified, and a line of stem cells homozygous for atleast one MHC allele present in the recipients cells is obtained from astem cell bank produced according to the disclosed methods. That line ofstem cells is then used to generate cells or tissue suitable fortransplant that are homozygous for at least one MHC allele present in.the recipients cells. The method of the present invention furthercomprises grafting the cells or tissue so obtained to the body of theperson in need of such a transplant. In a useful embodiment of theinvention, three, four, five, six or more of the MHC alleles of the lineof stem cells used to generate cells or tissue for transplant arehomozygous and match MHC alleles of the transplant recipient.

In a useful embodiment, the line of stem cells used to generate cells ortissue suitable for transplant is a line of totipotent or nearlytotipotent embryonic stem cells. In another useful embodiment, the lineof stem cells used to generate cells or tissue suitable for transplantis a line of hematopoietic stem cells. The lines of stem cells that canbe used to generate cells or tissue suitable for transplant areavailable “off the shelf” in the stem cell bank of the presentinvention. In a useful embodiment, the stem cell bank of the presentinvention comprises lines of totipotent, nearly totipotent, and/orpluripotent stem cells that are lines of rejuvenated, “hyper-youthfulcells” generated by nuclear transfer techniques. In another usefulembodiment, the stem cell bank of the present invention comprises one ormore lines of totipotent, nearly totipotent, and/or pluripotent stemcell having DNA that is genetically modified relative to the DNA of thehuman donor from which they are derived. For example, the inventioncomprises altering genomic DNA of the cells to replace a non-homozygousMHC allele with one that is homozygous, or to inhibit the effectivepresentation of a class I or class II HLA antigen on the cell surface,e.g., by preventing expression of β2-microglobulin, or by preventingexpression of one or more MHC alleles. Also, the invention encompassesintroducing one or more genetic modifications that result inlineage-defective stem cells, i.e., stem cells which cannotdifferentiate into specific cell lineages.

D. Methods for Making Stem Cell Lines with Homozygous MHC Alleles

Totipotent, nearly totipotent, and/or pluripotent stem cell lines thatmake up the stem cell banks of the present invention can be derived fromblastocyst embryos made up of cells that are homozygous for some or allof the histocompatibility antigen alleles, e.g., MHC alleles. Blastocystembryos useful for the present invention can be made by severaldifferent methods. In preferred embodiments of the invention, humanembryos are produced by fertilization, parthenogenesis, or by same orcross-species somatic cell nuclear transfer. In the case of humanembryos, for ethical reasons, they are never allowed to develop beyondthe stage of pre-implantation blastocysts of about 9-10 days before theinner cell mass cells are isolated and are cultured to produce embryonicstem (ES) cells. The cloning methods of the present invention whichutilize human embryos are restricted to human therapeutic cloningtechniques. The present invention does not include any methods thatpermit development of human embryos beyond the pre-implantation stage ofabout 9-10 days, nor does it include or contemplate reproductive cloningin any form.

Stem Cells from Embryos Produced by Union of Sperm and Egg

In one embodiment of the invention, human or non-human stem cells arederived from embryos produced in vitro by uniting sperm and eggs byknown means; for example, by in vitro fertilization (IVF) or byintracytoplasmic sperm injection (ICSI). To produce cells havinghomozygous MHC alleles, sperm and eggs can be obtained from individualsthat are closely related; e.g., brother and sister or one determined tohave similar MHC alleles. As in HLA typing for a transplant betweensiblings, there is about a 25% chance that an embryo produced withsibling's gametes will have matching HLA loci. The embryos produced byuniting sperm and eggs of related individuals are cultured in vitro toproduce early embryo including blastocysts from which ES cells or innercell masses are derived. HLA types of the resulting pluripotent celllines are determined by known means; e.g., by PCR, or by culturing asample of the cells under conditions that induce differentiation, andperforming serological testing of the cells using antibodies againstspecific HLA antigens. Pluripotent cell lines having one or morehomozygous MHC alleles are then selected for inclusion in the stem cellbank. Embryos produced by union of sperm and egg have normal geneticimprinting, i.e., they have the epigenetic contributions of both maleand female parents, so they develop to form blastocysts from whichpluripotent cells can be derived with high efficiency.

In the case where sperm and egg donors are not closely related sperm canbe banked from individuals with characterized MHC loci and used for IVFor ICSI fertilization of oocytes that also have characterized MHC locito produce embryos and stem cells with a high likelihood of generatinghomozygosity in the MHC loci.

Persons skilled in the art would recognize that the human embryosproduced by uniting sperm and eggs of closely related individualsaccording to the present invention may be viable and could be implantedinto human females to make pregnancies and develop to live births ofhumans having homozygous HLA alleles. This would be highly unethical, inview of the known risks to the health of the child that result fromclose inbreeding. As stated above, the present invention expressly doesnot comprise allowing the embryos to develop beyond blastocysts of about9-14 days.

Stem Cells Produced by Parthenogenesis

In another embodiment of the invention, totipotent and pluripotent humanstem cells are derived from embryos produced by parthenogenesis. Thestem cells obtained by this method are diploid, because extrusion of thesecond polar body following parthenogenetic activation is inhibited.Methods for producing a diploid human embryo by parthenogenesis, forculturing the embryo in vitro to form a blastocyst, and for culturingcells of the blastocyst to obtain stem cells, are described in co-ownedand co-pending PCT Application PCT/US02/37899 (Methods for Making andUsing Reprogrammed Human Somatic Cell Nuclei and Autologous and IsogenicStem Cells) filed Nov. 26, 2002, the disclosure of which is incorporatedherein by reference in its entirety. Similar methods for producingdiploid embryos by parthenogenesis using oocytes of rhesus monkeys andcynomolgus monkeys have been described by Mitalipov et al. (2001,Biology of Reproduction, 65:253-259) and Cibelli et al. (2002, Science,295:81), respectively, the contents of both of which are incorporatedherein by reference in their entirety.

In general, production of a diploid human embryo by parthenogenesiscomprises

-   -   a. obtaining oocytes from human donors induced to superovulate        by treatment with gonadotropins followed by hCG injection;    -   b. activating the oocytes at about 38-45 hours after hCG        stimulation;    -   c. exposing the activated oocytes to chemical treatment that        inhibits extrusion of the second polar body; and    -   d. culturing the embryo in vitro under conditions resulting in        formation of a blastocyst.

Oocyte activation is normally mediated by oscillations of intracellularCa+2 ion triggered by the sperm cell. Parthenogenetic activation of theoocytes can be achieved by any of the known means for inducing oocyteactivation. Such methods generally involve exposing the oocyte toethanol, electroporation, calcium ionophore, ionomycin, inositol1,4,5-triphosphate to increase the intracellular Ca⁺² ion concentrationin the oocyte, in combination with a treatment that temporarily inhibitsprotein synthesis or protein phosphorylation. For example, Mitalipov etal. (supra, p. 254) describe two such methods that result in productionof diploid parthenogenetic blastocysts from oocytes of rhesus monkeys.In one method, the oocytes are incubated briefly in medium containingionomycin and calcium, followed by incubation for several hours inmedium containing 6-aminomethylpurine (DMAP), an inhibitor of proteinphosphorylation. In the other method, the oocytes are electroporatedthree times in medium containing calcium, and between eachelectroporation, the oocytes are incubated for about 30 minutes inmedium containing cycloheximide, an inhibitor of protein synthesis, andcytochalasin B, an inhibitor of microfilament synthesis.

Using a similar method Cibelli et al. (supra) parthenogeneticallyactivated oocytes of a cynomolgus monkey; cultured the activated oocytesin vitro to produce a diploid blastocysts; and isolated a line ofdiploid ES cells from cells of the inner cell mass of aparthenogenesis-derived embryo; and showed that the ES cells are capableof differentiating into cell types of all three embryonic germ layers.This is also described in U.S. Ser. No. 09/697,297 by Cibelli et al,which is incorporated by reference in its entirety here.

Oocytes are obtained from women having MHC alleles of the type neededfor the stem cell bank. The oocytes are parthenogenetically activatedand are cultured to form blastocysts. Using known methods, the innercell mass cells of the blastocysts are cultured in vitro to generatediploid embryonic stem cells. Because extrusion of the second polar bodyafter meiosis II was prevented, the homologous chromosomes of such EScells are actually the sister chromatids that were joined together as adyad during meiosis I. Since the sister chromatids were formed byreplication of a single set of chromosomes at the outset of meiosis,they will have identical DNA sequences, except for those regions thatwere exchanged with the homologous dyad during the recombination stageof meiosis. The HLA genes of the MHC are tightly linked, andrecombination in this region is rare occurring with a frequency of about1%. The two sets of homozygous HLA alleles in theparthenogenetically-derived stem cell lines obtained with oocytes from agiven donor reflect the HLA haplotypes of the maternal and paternalcopies of chromosome 6 that the donor inherited from her parents. Knownscreening methods can be performed to identify the cell lines that havenon-homozygous HLA antigens due to genetic recombination, and toidentify the homozygous HLA alleles of each stem cell line.

Stem Cells Produced by Haploidization

In another embodiment of the invention, totipotent and pluripotent humanstem cells are derived from embryos produced by union of two haploidsthat are homozygous for one or more MHC alleles.

Methods for producing embryos by fusion of two haploid genomes aredescribed in U.S. Ser. No. 10/344,724, filed on Feb. 14, 2003 entitled,“Use of Haploid Genomes for Genetic Diagnosis, Modification andMultiplication,” which is incorporated by reference in its entiretyherein.

A bank of stem cell lines according to the present invention can be.obtained by screening the population- and -identifying individualshaving cells which express desired MHC antigens, and obtaining donationsof the somatic cells from these individuals. However, individuals thatare homozygous for MHC antigens are rare, because they are only found ininbred population. Thus, the useful embodiment of the invention isutilization of heterozygous donor cells to create homozygous stem cells.

In this method, somatic cells are introduced into enucleated humanoocytes, and the newly constructed oocytes are activated to inducehaploidization (Tesarik et al., 2001 R B Online. 2:160-164),Lachem-Kaplan et al, 2001 R B Online 3: 205-211. When a protocol forprimate oocyte activation are used, approximately 90% of eggs yieldpseudo-polar body (Shoukhrat et al, 2001 Biol Reprod 65:253-259). Thesepseudo-polar bodies a re used for genotyping using well establishedtechniques. Other haploid embryos also can be constructed bytransferring cells from other donors using the same protocol. Or thedonor oocytes can be screened for the presence of desired MHC alleleafter activation to generate haploid oocytes. Screening of the firstpolar bodies will reveal the genotype of the oocytes as in above thereconstructed eggs. The activation can be done chemically and/or byinjecting sperm factors (see U.S. Application No. 60/191,089 of RafaelFissore filed Mar. 22, 2000 incorporated by reference in its entiretyherein) easily unless 2nd polar body extrusion is blocked systematically(incorporated by reference in its entirety herein). The remainingpronuclei are transferred to construct diploid embryos by pronucleartransfer techniques. These techniques have been well established andused widely in developmental biology fields for more than a decade. Toavoid possible imprinting disturbance, morula stage human embryo lysatesare injected into the newly constructed eggs. These embryo lysates areknown to have ability to modify imprinting status of murine androgenoneso effectively to make live born animals, otherwise develop very poorlyin vitro and died out after implantation (Hagemman and First, 1992Development 114:997-1001).

More particularly, the invention includes methods for generating stemcells by haploidization comprising the steps of:

-   -   a. Inserting a somatic donor cell, or the nucleus of such a        cell, into an oocyte that is free of oocyte genomic DNA.    -   b. Activation of the reconstructed embryos to expel haploidal        genome into a pseudopolar body.    -   c. Screening of the pseudopolar body for the genotyping of        remaining pronucleus.    -   d. Union of the two pronuclei to generate diploid embryos by        pronuclei transfer. Or alternatively, transferring a pronucleus        to an activated haploid oocyte which has desired haploid genome    -   e. Injection of human morular stage embryo lysates to the        reconstructed embryos.    -   f. Culturing embryo and generating stem cells/or differentiated        cells or tissue needed for transplant from cells of said        embryos.

In addition, haploid genomes can be derived by other means known in theart, including the use of the first and second polar bodies. Whileoccasionally, such DNA is fragmented, intact genomes can be obtained asevidenced by the production of live mice from polar body DNA (Wakayama,T., and Yanagimachi, R. Biol. Reprod. 1998. 59(1) 100-4) and thesehaploid or diploid genomes can be used as described above.

Stem Cells Produced by Cytoplasm Transfer

Totipotent and pluripotent stem cells homozygous for histocompatibilityantigens, e.g., MHC antigens can also be produced by transferringcytoplasm from an oocyte or an ES cell into a somatic cell that ishomozygous for MHC antigens, so that the chromatin of the somatic cellis reprogrammed and the somatic cell de-differentiates to generate apluripotent or totipotent stem cell. Methods for convertingdifferentiated cells into de-differentiated, pluripotent, stem orstem-like cells that can be induced to re-differentiate into a cell typeother than that of the initial differentiated cells, are described inco-owned and co-pending U.S. application Ser. No. 09/736,268, filed Dec.15, 2000, and U.S. application Ser. No. 10/112,939 filed Apr. 2, 2002,both by Karen B. Chapman, the disclosures of both of which areincorporated herein by reference in their entirety.

Stem Cells from Embryos Produced by Nuclear Transfer

In another embodiment of the invention, totipotent, nearly totipotent,and/or pluripotent human stem cells that are homozygous for one or moreMHC alleles are derived from embryos produced in vitro by somatic cellnuclear transfer techniques. The totipotent and/or pluripotent stemcells generated by this embodiment of the invention will have thegenomic DNA of the somatic donor cell used for nuclear transfer. Whenthe somatic donor cell is homozygous for an MHC allele, the stem cellsgenerated by nuclear transfer cloning will also be homozygous for theMHC allele.

A bank of stem cell lines according to the present invention can beobtained by screening a species, preferably human population andidentifying individuals that are homozygous for clinical MHC antigens,and obtaining donations of somatic cells from these individuals.Individuals having homozygous MHC alleles are often found in inbredpopulations. Alternatively, somatic cells, preferably human, homozygousfor MHC loci that are useful for the present invention can be producedby obtaining somatic cells that are heterozygous for an MHC allele, andgenetically altering the DNA of the cells using known methods so thatthey are homozygous for one or more MHC loci. This can be done, forexample, by using well-known homologous recombination techniques toreplace a non-homozygous MHC allele with one that is homozygous.

In a useful embodiment of the invention, donors of somatic cells to beused in nuclear transfer according to the present invention may beselected to provide cells that are relatively resistant to blood cellcancers, for use in reconstituting the blood of blood cancer patients.Such blood cells can be chosen based on their natural killer (NK) cellphenotype. The somatic cell donors who having resistance to blood cellcancers can be selected to have homozygous MHC alleles, or the donatedcells can be genetically altered to have one or more homozygous MHCalleles as discussed above.

The donated cells are cloned by nuclear transfer techniques that resultin production of blastocyst embryos from which are obtained totipotentand/or pluripotent stem cells that are homozygous for one or more MHCloci. For each cell line to be produced, a somatic donor cell that ishomozygous for a MHC allele, or the nucleus or set of chromosomes ofsuch a cell, is inserted into a human oocyte that is coordinatelyenucleated to produce a nuclear transfer unit that develops as anembryo. The embryo is cultured ex vivo to the blastocyst stage, andtotipotent and/or pluripotent stem cells are derived from inner cellmass (ICM) cells of the embryo that have the genomic DNA of the donorcell. In a useful embodiment, the stem cell bank comprises totipotent,nearly totipotent ES cells homozygous for MHC antigens. Totipotent andpluripotent stem cells homozygous for various combinations of MHCantigens are assembled and maintained as a bank of cells available fortherapeutic transplantation.

Methods for transferring the nuclear DNA of a somatic cell of a patientinto an oocyte to effect the reprogramming of the chromatin and producean NT unit from which are generated pluripotent stem cells andtotipotent ES cells are described, for example, in co-owned andco-pending U.S. application Ser. No. 09/655,815 filed Sep. 6, 2000; andU.S. application Ser. No. 09/797,684 filed Mar. 5, 2001; and also in PCTApplication No. PCT/US02/37899 (Methods for Making and UsingReprogrammed Human Somatic Cell Nuclei and Autologous and Isogenic StemCells) filed Nov. 26, 2002, the disclosures of all three of which areincorporated herein by reference in their entirety. Similar methods aredescribed in co-owned and co-pending U.S. application Ser. No.09/527,026 filed Mar. 16, 2000, Ser. No. 09/520,879 filed Apr. 5, 2000,and Ser. No. 09/656,173 filed Sep. 6, 2000, the disclosures of which areincorporated herein by reference in their entirety. In general, methodsfor cloning by somatic cell nuclear transfer to produce stem cells forgenerating cells or tissue useful for transplantation comprise the stepsof:

-   -   a. inserting a somatic donor cell, or the nucleus of such a        cell, into an oocyte and removing the oocyte genomic DNA        (enucleation) under conditions that produce an activated nuclear        transfer unit that develops as an embryo; and    -   b. generating stem cells and/or differentiated cells or tissue        needed for transplant from cells of said embryo.

Such a method can be used to generate lines of totipotent or nearlytotipotent ES cells that can be cultured under conditions in which theydifferentiate into specific, recognized cell types. Such ES cells havethe capacity to differentiate into every cell type of the body,including the germ cells. The stem cells produced by somatic cellnuclear transfer have the patients genomic DNA, so the differentiatedcells and tissues generated from such stem cells are nearly completelyautologous—all of the cells' proteins, are encoded by the patients ownDNA except for those proteins encoded by the cells' mitochondria, whichderive from the oocyte. Accordingly, differentiated cells and tissuesgenerated from stem cells produced by nuclear transfer methods can betransplanted to the person who provided the nuclear donor cell withouttriggering the severe rejection response that results when foreign cellsor tissue are transplanted.

As described in the above-identified co-pending applications, thesomatic donor cell used for nuclear transfer to produce human stem cellshomozygous for a MHC allele according to the present invention can be ofany. somatic cell type in the body. For example, the somatic donor cellcan be a cell selected from the group consisting of fibroblasts. Bcells, T cells, dendritic cells, keratinocytes, adipose cells,epithelial cells, epidermal cells, chondrocytes, cumulus cells, neuralcells, glial cells, astrocytes, cardiac cells, esophageal cells, musclecells, melanocytes, hematopoietic cells, macrophages, monocytes, andmononuclear cells. The somatic donor cell can be obtained from any organor tissue in the body; for example, it can be a cell from an organselected from the group consisting of liver, stomach, intestines, lung,stomach, intestines, lung, pancreas, cornea, skin, gallbladder, ovary,testes, kidneys, heart, bladder, and urethra.

Methods for generating rejuvenated, “hyper-youthful” stem cells anddifferentiated somatic cells having the genomic DNA of a human somaticdonor cell are described in co-owned and co-pending U.S. applicationSer. No. 09/527,026 filed Mar. 16, 2000, Ser. No. 09/520,879 filed Apr.5, 2000, and Ser. No. 09/656,173 filed Sep. 6, 2000, the disclosures ofwhich have been incorporated herein by reference in their entirety. Forexample, rejuvenated, “hyper-youthful” stem cells having the genomic DNAof a human somatic cell donor can be produced by a method comprising:

-   -   a. isolating normal, somatic cells from a human donor, and        passaging or otherwise inducing the cells into a state of        checkpoint-arrest, senescence, or near-senescence,    -   b. transferring a checkpoint-arrested, senescent, or        near-senescent donor cell, the nucleus of said cell, or        chromosomes of said cell, into a recipient oocyte, and        coordinately removing the oocyte genomic DNA from the oocyte, to        generate an embryo; and    -   c. generating rejuvenated stem cells from said embryo having the        genomic DNA of the donor cell.

As described in the above-identified co-pending applications, thepluripotent and totipotent stem cells homozygous for a MHC allele of thepresent invention that are produced by nuclear transfer using acheckpoint-arrested, senescent, or near-senescent donor cell arerejuvenated cells that are distinguished from other cells in havingtelomeres that are longer than the corresponding telomeres of thecheckpoint-arrested, senescent, or near-senescent donor cell. Moreover,the telomeres of such rejuvenated cells are on average at least as longas the telomeres of age-matched control cells of the same type andspecies that are not generated by nuclear transfer techniques. Inaddition, the nucleotide sequences of the tandem (TTAGGG)_(n) repeatsthat comprise the telomeres of such rejuvenated cells are more uniformand regular; i.e., have significantly fewer non-telomeric nucleotidesequences, than are present in the telomeres of age-matched controlcells of the same type and species that are not generated by nucleartransfer. Such rejuvenated cells are “hyper-youthful”, in that theproliferative life-span of the rejuvenated cells is at least as long as,and is typically longer than, the proliferative life-span of age-matchedcontrol cells of the same type and species that are not generated bynuclear transfer techniques. Such rejuvenated cells also have patternsof gene expression that are characteristic of youthful cells; forexample, activities of EPC-1 and telomerase in such rejuvenated cellsare typically greater than EPC-1 and telomerase activities inage-matched control cells of the same type and species that are notgenerated by nuclear transfer techniques.

As described in the above-identified co-pending applications,rejuvenated totipotent and/or pluripotent stem cells can be generatedfrom an embryo produced by nuclear transfer by methods comprisingobtaining a blastocyst, an embryonic disc cell, inner cell mass cell, ora teratoma cell using said embryo, and generating the pluripotent and/ortotipotent stem cells from said blastocyst, inner cell mass cell,embryonic disc cell, or teratoma cell.

As described in co-owned and co-pending U.S. application Ser. No.09/685,061 filed Oct. 6, 2000, Ser. No. 09/809,018 filed Mar. 16, 2001,and Ser. No. 09/874,040 filed Jun. 6, 2001, the recipient oocyte may bederived from a non-human mammal. For example, the oocyte may be from amammal selected from the group consisting of sheep, bovines, bovines,pigs, horses, rabbits, guinea pigs, mice, hamsters, rats, and non-humanprimates. In a preferred embodiment, the oocyte is from a bovine mammal,or a rabbit. A stem cell line having the genome of a human cell that isderived using a nonhuman oocyte is referred to herein as a “human” stemcell line, even though the mitochondria of such cells are of a non-humantype.

Genetically Modified Stem Cells

The methods of the present invention include producing totipotent and/orpluripotent stem cells homozygous for MHC antigens that are geneticallymodified relative to the cells of the human donor from which they wereoriginally obtained. The stem cells can be genetically modified in anymanner that enhances or improves the overall efficiency by which cellsfor transplant are produced and the therapeutic efficacy of the celltransplantation. Methods that use recombinant DNA techniques tointroduce modifications at selected sites in the genomic DNA of culturedcells are well known. Such methods can include (1) inserting a DNAsequence from another organism (human or non-human) into target nuclearDNA, (2) deleting one or more DNA sequences from target nuclear DNA, and(3) introducing one or more base mutations (e.g., site-directedmutations) into target nuclear DNA. Such methods are described, forexample, in Molecular Cloning, a Laboratory Manual, 2nd Ed., 1989,Sambrook, Fritsch, and Maniatis, Cold Spring Harbor Laboratory Press;U.S. Pat. No. 5,633,067, “Method of Producing a Transgenic Bovine orTransgenic Bovine Embryo,” DeBoer et al., issued May 27, 1997; U.S. Pat.No. 5,612,205, “Homologous Recombination in Mammalian Cells,” Kay etal., issued Mar. 18, 1997; and PCT publication WO 93/22432, “Method forIdentifying Transgenic Pre-Implantation Embryos,” all of which areincorporated by reference herein in their entirety. Such methods includetechniques for transfecting cells with foreign DNA fragments and theproper design of the foreign DNA fragments such that they effectinsertion, deletion, and/or mutation of the target DNA genome. Forexample, known methods for genetically altering cells that usehomologous recombination can be used to insert, delete, or rearrange DNAsequences in the genome of a cell of the present invention. A geneticsystem that uses homologous recombination to modify targeted DNAsequences in a mammalian cell to “knock-out” a cell's ability to expressa selected gene is disclosed by Capecchi et al. in U.S. Pat. Nos.5,631,153 and 5,464,764, the contents of which are incorporated hereinin their entirety. Such known methods can be used to insert into thegenomic DNA of a cell an additional (exogenous) DNA sequence comprisingan expression construct containing a gene that is to be expressed in themodified cell. The gene to be expressed can be operably linked to any ofa wide variety of different types of transcriptional regulatorysequences that regulate expression of the gene in the modified cell. Forexample, the gene can be under control of a promoter that isconstitutively active in many different cell types, or one that isdevelopmentally regulated and is only active in one or a few specificcell types. Alternatively, the gene can be operably linked to aninducible promoter that can be activated by exposure of the cell to aphysical (e.g., cold, heat, light, radiation) or chemical signal. Manysuch inducible promoters and methods for using them effectively are wellknown. Examples of the characteristics and use of such promoters, and ofother well-known transcriptional regulatory elements such as enhancers,insulators, and repressors, are described, for example, in TransgenicAnimals, Generation and Use, 1997, edited by L. M. Houdebine, HardwoodAcademic Publishers, Australia, the contents of which are incorporatedherein by reference.

Stem cells homozygous for MI-IC antigens that have multiple geneticalterations can be produced using known methods. For example, one canproduce cells that are modified at multiple loci, or cells that aremodified at a single locus by complex genetic alterations requiringmultiple manipulations. To produce stem cells having multiple geneticalterations, it is useful to perform the genetic manipulations onsomatic cells cultured in vitro, and then to clone the geneticallyaltered cells by somatic cell nuclear transfer and generate ES cellshaving multiple genetic alterations from the resulting blastocysts.Methods for generating genetically modified cells using nuclear transfercloning techniques are described, for example, in co-owned andco-pending U.S. application Ser. No. 09/527,026 filed Mar. 16, 2000,Ser. No. 09/520,879 filed Apr. 5, 2000, and Ser. No. 09/656,173 filedSep. 6, 2000, the disclosures of which have been incorporated herein byreference in their entirety.

Alternatively, the totipotent and/or pluripotent stem cells havinghomozygous MHC alleles that are produced by any of the methods describedabove can be genetically modified directly using known methods. Forexample, Zwaka et al. have described a method for genetically modifyinghuman ES cells in vitro by homologous recombination (NatureBiotechnology, Vol. 21, No. 3, March, 2003).

In generating stem cells by nuclear transfer, it is useful togenetically modify the nuclear donor cell to enhance the efficiency ofembryonic development and the generation of ES cells. The gene productsof the Ped type, which are members of the Class I MHC family and includethe Q7 and Q9 genes, are reported to enhance the rate of embryonicdevelopment. Modification of the DNA of nuclear donor cells by insertionof DNA expression constructs that provide for the expression of thesegenes, or their human counterparts, will give rise to nuclear transferembryos that grow more quickly. It appears that these genes are onlyexpressed early in blastocyst development and so are not expected to bedisruptive of later development.

The efficiency of embryonic development can also be enhanced bygenetically modifying the nuclear donor cell to have increasedresistance to apoptosis. Genes that induce apoptosis are reportedlyexpressed in preimplantation stage embryos (Adams et al, Science,281(5381):1322-1326 (1998). Such genes include Bad, Bok, BH3, Bik, Hrk,BNIP3, BimL, Bad, Bid, and EGL-1. By contrast, genes that reportedlyprotect cells from programmed cell death include BcL-XL, Bcl-w, Mcl-1,A1, Nr-13, BHRF-1, LMW5-HL, ORF16, Ks-Bel-2, E1B-19K, and CED-9. Nucleardonor cells can be constructed in which genes that induce apoptosis are“knocked out” or in which the expression of genes that protect the cellsfrom apoptosis is enhanced or turned on during embryonic development.Expression constructs that direct synthesis of antisense RNAs orribozymes that specifically inhibit expression of genes that induceapoptosis during early embryonic development can also be inserted intothe DNA of nuclear donor cells to enhance development of nucleartransfer-derived embryos. Apoptosis genes that may be expressed in theantisense orientation include BAX, Apaf-1, and capsases. Many DNAs thatpromote or inhibit apoptosis have been reported and are the subject ofnumerous patents. The construction and selection of genes that affectapoptosis, and of cell lines that express such genes, is disclosed inU.S. Pat. No. 5,646,008, the contents of which are incorporated hereinby reference.

Stem cells can be produced that are genetically modified grow moreefficiently in tissue culture than unmodified cells; e.g., by increasingthe number of growth factor receptors on the cells' surface. Use of stemcells having such modifications reduces the time required to generate anamount of cells for transplant that is sufficient to have therapeuticeffect.

The histocompatibility of a line of cells to be used for transplant witha patient in need of such as transplant may be increased by altering thegenomic DNA of the cells to replace a non-homozygous MHC allele with onethat is homozygous and matches an HLA allele of the patient.Alternatively, the genomic DNA of the cells can be modified to inhibitthe effective presentation of a class 1 or class II HLA antigen on thecell's surface; for example, by introducing a genetic alteration thatprevents expression of β2-microglobulin, which is an essential componentof class I HLA antigens; by introducing genetic alterations in thepromoter regions of the class I and/or or class II MHC genes; or simplyby deleting a portion of the DNA of one or more of the class I and/or orclass II MHC genes sufficient to prevent expression of the gene(s).

Stem cells of the invention can be genetically modified (e.g., byhomologous recombination) to have a heterozygous knock-out of the Id1gene, and a homozygous knockout of the Id3 gene. As described inco-owned and co-pending PCT Application No. PCT/USU3/01827 (StemCell-Derived Endothelial Cells Modified to Disrupt Tumor Angiogenesis),filed Jan. 22, 2003, these stem cells can be induced to differentiateinto Id1+/−, M3−/− endothelial cell precursor cells that are useful forthe treatment of cancer because they give rise to endothelial cells thatdisrupt and inhibit tumor angiogenesis.

Stem cells of the invention can also be genetically modified to providea therapeutic gene product that the patient requires, e.g., due to aninborn error of metabolism. Many genetic diseases are known to resultfrom an inability of a patient's cells to produce a specific geneproduct. The present invention making genetically altered stem cellsthat can be used to produce cells with homozygous MHC alleles fortransplantation that are genetically modified to synthesize enhancedamounts of a gene product required by the transplant recipient. Forexample, hematopoietic stem cells that are genetically altered toproduce and secrete adenosine deaminase can be prepared for transplantto a patient suffering from adenosine deaminase deficiency. The methodsof the present invention permit production of such cells without the useof recombinant retrovirus, which can insert at a site in the genomic DNAthat disrupts normal growth control and causes neoplastictransformation.

Stem cells of the invention can also be genetically modified byintroduction of a gene that causes the cell to die. The gene can be putunder control of in inducible promoter. If for any reason thetransplanted cells react in a in a way that can harm the recipient,expression of the suicide genes can be induced to kill the transplantedcells. Use of inducible suicide genes in this manner is known in theart. Suitable suicide genes include genes encoding HSV thymidine kinaseand cytodine deaminase, with which cell death is induced by gancyclovirand 5-fluorocytosine, respectively.

The cells may be modified to knockout one or more histocompatibilityantigen alletes, e.g., MHC alleles such that only one set remains. Thisleads to an underexpression of the MHC genes, but a phenotype effectivein reducing the complexity of the MHC serotype and effective inproducing cells capable of otherwise functioning and useful in thetreatment of disease. Alternatively, homozygosity can be engineered intothe cell lines by the targeted introduction of the appropriate allelesto the nonhomologous set, to result in homozygosity. In addition, thechromosome carrying the MHC genes can be removed from cells by laserablation and a chromosome carrying the identical chromosome as remainsin the cell can be added by microsome-mediated chromosome transfer, orby other techniques known in the art.

The present invention is by no means limited to the foregoing examplesof genetic alterations. Persons skilled in the art will be able toidentify numerous other ways by which stem cells produced according tothe present invention can be genetically modified to enhance theirutility.

Preparing Totipotent and/or Pluripotent Stem Cells

Stem cells are present in the earliest stages of embryo formation.Embryonic stem cells (ES cells) are undifferentiated stem cells that arederived from the inner cell mass (ICM) of a blastocyst embryo.Totipotent and/or nearly totipotent ES cell lines can be derived fromhuman blastocysts using known methods comprising removing cells of theinner cell mass of an early blastocyst by microsurgery or immuno-surgeryand culturing the cells in vitro (e.g., see U.S. Pat. No. 6,235,970, thecontents of which are incorporated herein by reference in theirentirety). For example, such methods are described in co-owned andco-pending PC7 application, PCT/US02/37899 (Methods for Making and UsingReprogrammed Human Somatic Cell Nuclei and Autologous and Isogenic StemCells) filed Nov. 26, 2002, using blastocysts produced both by nucleartransfer and by parthenogenesis, the disclosure of which areincorporated herein by reference in its entirety. Thomson et al. alsodescribes methods by which ES cell lines can be derived fromprimate/human blastocysts (Science, 1988, 282:1145-1147; and Proc. Natl.Acad. Sci., USA, 1995, 92:7544-7848), which are incorporated byreference herein in their entirety. A detailed .method for preparinghuman ES cells is also described in Thomson's U.S. Pat. No. 6,200,806,“Primate Embryonic Cells,” issued Mar. 13, 2001, the disclosure of whichis incorporated herein by reference in its entirety. As describedtherein, a human ES cell line can be derived from cells of a blastocystby a method comprising:

-   -   a. isolating a human blastocyst;    -   b. isolating cells from the inner cell mass of the blastocyst;    -   c. plating the inner cell mass cells on embryonic fibroblasts so        that inner-cell mass-derived cell masses are formed;    -   d. dissociating the mass into dissociated cells;    -   e. replating the dissociated cells on embryonic feeder cells;    -   f. selecting colonies with compact morphologies and cells with        high nucleus to cytoplasm ratios and prominent nucleoli; and    -   g. culturing the selected cells to generate a pluripotent human        embryonic stem cell line.

Methods for growing human ES cells and maintaining them in anundifferentiated state without culturing them on a layer of feeder cellshave also been described (Xu et al., Nature Biotechnology, 2001,19:971-4, the contents of which are incorporated herein by reference intheir entirety). Feeder-free culture of stem cells can reduce the riskof contamination of the cells by pathogens that may reside in the feedercells.

Generating Differentiated Cells

Stem cells are widely regarded as an abundant source of pluripotentcellular material that can be directed to differentiate into cells andtissues that are suitable for transplantation into patients in need ofsuch cell and tissue transplants. ES cells appear to have unlimitedproliferative potential and are capable of differentiating into all ofthe specialized cell types of a mammal, including the three embryonicgerm layers (endoderm, mesoderm, and ectoderm), and all somatic celllineages and the germ line. Using known methods, totipotent or nearlytotipotent ES cells can be cultured under conditions in which theydifferentiate into pluripotent or multipotent stem cells such ashematopoietic or neuronal stem cells. Alternatively, totipotent ES cellscan be cultured under conditions in which they differentiate into aterminally differentiated cell type such as a cardiac muscle cell.Totipotent and/or pluripotent stem cells homozygous for MHC allelesproduced by the methods of the present invention can be cultured usingmethods and conditions known in the art to generate cell lineages thatdifferentiate into many, if not all, of the cell types of the body, fortransplant into human patients in need of such transplants. Such stemcells having one or more homozygous MHC alleles can differentiate intocells selected from the group consisting of immune cells, neurons,skeletal myoblasts, smooth muscle cells, cardiac muscle cells, skincells, pancreatic islet cells, hematopoietic cells, kidney cells, andhepatocytes. For example, methods have been described by whichtotipotent or nearly totipotent ES cells are induced to differentiate invitro into cardiomyocytes (Paquin et al., Proc. Nat. Acad. Sci. (2002)99:95509555), hematopoietic cells (Weiss et al., Hematol. Oncol. Clin.N. Amer. (1997) 11(6): 1185-98; also U.S. Pat. No. 6,280,718),insulin-secreting beta cells (Assady et al., Diabetes (2001)50(8):1691-1697), and neural progenitors capable of differentiating intoastrocytes, oligodendrocytes, and mature neurons (Reubinoff et al.,Nature Biotechnology (2001) 19:1134-1140; also U.S. Pat. No. 5,851,832).

Novel screening methods that make use of gene trapped cell lines andprovide means for efficiently identifying combinations of biological,biochemical, and physical agents or conditions that induce stem cells todifferentiate into cell types useful for transplant therapy, and forpreparing and isolating specific differentiated cell types, aredescribed in co-owned and co-pending U.S. application Ser. No.10/227,282, filed Aug. 26, 2002, and in U.S. Provisional Application No.60/418,333 (“Methods Using Gene Trapped Stem Cells for Marking Pathwaysof Stem Cell Differentiation And Making and Isolating DifferentiatedCells”), filed Oct. 16, 2002, the contents of both of which are alsoincorporated herein by reference in their entirety.

In a useful embodiment of the present invention, a stem cell bank isproduced that comprises hematopoietic stem cells homozygous for MHCantigens. A method for inducing the differentiation of pluripotent humanembryonic stem cells into hematopoietic cells useful for transplantaccording to the present invention is described in U.S. Pat. No.6,280,718, “Hematopoietic Differentiation of Human Pluripotent EmbryonicStem Cells,” issued to Kaufman et al. on Aug. 28, 2001, the disclosureof which is incorporated herein by reference in its entirety. The methoddisclosed in the patent of Kaufman et al. comprises exposing a cultureof pluripotent human embryonic stem cells to mammalian hematopoieticstromal cells to induce differentiation of at least some of the stemcells to form hematopoietic cells that form hematopoietic cell colonyforming units when placed in methylcellulose culture.

Those skilled in the art will appreciate that, using currently availablemethodologies, the totipotent and pluripotent stem cells of the presentinvention can also be used to generate tissues formed of two or moredifferent cell types homozygous for a MHC allele, for transplant to aperson in need of such a tissue transplant.

The pluripotent and totipotent stem cells homozygous for MHC antigensthat are generated according to the present invention, and the lines ofdifferentiated cells obtained from these stem cells, are produced andisolated under Good Manufacturing Practices (GMP) conditions.

Providing Histocompatible Transplants to People Needing them

The methods for generating stem cells and differentiated cells havinghomozygous MHC alleles described above provide effective solutions tomany of the problems associated with obtaining cells for transplant thatare histocompatible with a transplant recipient. However, de novoproduction of histocompatible cells and tissue for transplantation by invitro fertilization, parthenogenesis, or nuclear-transfer-based methodsfor each patient in need of transplant is time-consuming. The timerequired to prepare “customized” cells or tissue for transplantationhaving the same HLA antigens as the transplant recipient can beproblematic when the health of the would-be recipient is rapidlydeteriorating for want of a transplant. Therefore, one or more of theabove-described methods for generating stem cells and differentiatedcells having homozygous MHC alleles are used to produce a stem cell bankcomprising many different lines of stem cells, each having a differentcombination of homozygous MHC alleles present in the population. When apatient is found to be in need of a particular type of cell transplant,a line of stem cells from the stem cell bank having homozygous MHCalleles matching those of the patient can be taken “off the shelf” andcultured under conditions causing them to differentiate into the type(s)of cells needed. The differentiated cells are then isolated using knownmethods, and are provided to the patients physician for transplant.

Accordingly, the present invention includes the process of identifyingthe type of cells needed for transplant, and the blood type and HLAantigens of the transplant recipient, selecting stem cells from the stemcell bank that differentiate into the cell type needed and havehomozygous HLA antigens that match those of the transplant recipient;culturing the stem cells under conditions in which they differentiateinto the cell type needed; isolating the differentiated cells needed fortransplant; and providing these to the patient's physician fortransplant into the patient.

The differentiated cells for transplant produced by these methods arehomozygous for at least one HLA antigen present on cells of thetransplant recipient. Histocompatibility of the cells for transplant andthe recipient increases as a function of the number of homozygous HLAantigens of the cells for transplant that match HLA antigens of therecipient. The greater the number of homozygous HLA antigens of thecells for transplant that match HLA antigens of the recipient, thelonger the graft is expected to survive without being rejected. Thecells for transplant provided by the invention will therefore have one,two, three, four, five, six, or more homozygous HLA antigens that matchHLA antigens of the recipient. For example, cells for transplantproduced by the present invention can have homozygous HLA-A, HLA-B, andHLA-DR antigens that match HLA antigens of the recipient. Alternatively,the cells for transplant produced by the present invention can havehomozygous HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, and HLA-DP antigens thatmatch HLA antigens of the recipient. The ability to select stem cells“off the shelf to produce cells for transplant having a relatively highnumber of homozygous HLA antigens that match those of a prospectivetransplant recipient depends on the size and complexity of the stemcells bank. A stem cell bank containing from 100,000 to 200,000different stem cell lines, each having a different combination ofhomozygous HLA-A, HLA-B, and HLA-DR antigens, is required in order to beable to provide cells with homozygous HLA-A. HLA-B, and HLA-DR antigensthat match the corresponding HLA antigens of a large percentage ofpeople in a diverse population such as that of North America. Use ofcells from individuals with blood type 0 can avoid rejection based onABO blood type; but there would have to be two versions of each celltype in the stem cell bank in order to provide matches to the Rh(+) andRh(−) blood types. Accordingly, a stem cell bank containing severalhundred thousand stem cell lines can be expected to provide “off theshelf stem cells that can be used to generate differentiated cellsneeded for transplant that have homozygous HLA-A, HLA-B, and HLA-DRantigens matching those of a person in need of such a transplant.

The stem cell bank of the present invention contains lines oftotipotent, nearly totipotent, and/or pluripotent human stem cells, eachhaving a specific combination of one or more homozygous HLA antigens.The lines of stem cells that can be used to generate cells or tissuesuitable for transplant can be lines of totipotent or nearly totipotenthuman ES cells. The stem cell lines can also be pluripotent, partiallydifferentiated stem cells such as myoblasts, hematopoietic stem cells,neuronal precursor cells, and endothelial cell precursor cells.

Therapeutic Cell Transplantation

Using the methods of the present invention, a line of totipotent orpluripotent stem cells can be selected from a bank of such stem cellsthat are homozygous for one or more histocompatibility antigen alleles,in the case of human stem cells. MHC alleles that match an MHC allele ofa patient in need of transplant. For example, the stem cells can havehomozygous HLA-A, HLA-B, and HLA-DR alleles that match HLA-A, HLA-B, andHLA-DR alleles of the patient. The stem cells are cultured ex vivo underconditions in which they are induced to differentiate into partially orfully differentiated cell types that are suitable for transplant andhave homozygous MHC alleles that match MHC alleles of the patient inneed of the transplant.

The partially or fully differentiated cells needed for transplant areisolated from other cell types, e.g., using antibody-based separationmethods such as cell sorting or immunomagnetic beads, and antibodiesthat are specific for one or more differentiation antigens on thesurface of the cell type needed for transplant, as described in U.S.Provisional Application No. 60/418,333, filed Oct. 16, 2002, thedisclosure of which is incorporated herein by reference in its entirety.The isolated partially or fully differentiated cells are thenadministered by transplantation to the patient using known methods.Methods for transplantation of epidermal cells, hematopoietic stemcells. Islet of Langerhans cells, chondrocytes, hepatocytes, myoblasts,neural cells, and endothelial cells are reviewed by Inverardi et al.(Transplantation Biology, Cellular and Molecular Aspects, Chapter 56,1996, ed. by Tilney et al., Lippincott-Raven, Philadelphia, Pa. Themethod to be used to transplant or engraft cells to a patient isrecognized as depending on the type of cells to be transplanted, and onthe pathology of the patient.

Cells Homozygous for Recessive Disease-Causing Genes

Recessive alleles responsible for genetically inherited diseases areendemic in the population. If cells of people carrying a recessivedisease-causing gene are used to produce stem cells having homozygousHLA alleles from embryos generated by parthenogenesis, or with sperm andeggs of closely related individuals, there is a relatively highlikelihood that some of the stem cell lines obtained also be homozygousfor the recessive disease-causing gene. The stem cell lines produced bythe methods of the present invention can therefore be screened toidentify those which are homozygous for a recessive disease-causinggene. Such screening can be carried out using known methods. Forexample, DNA sequences of the cells can be amplified by the polymerasechain reaction (PCR) and analyzed by DNA sequencing, restriction enzymecleavage, or by hybridization to an array of oligomers, e.g., on amicrochip. Examples of recessive-disease causing genes to be screenedfor include, but are not limited to, of recessive genes causing thefollowing conditions:

Adenosine deaminase deficiency

Albinism

Adenylosuccinate lyase deficiency

Alpha-1 antitrypsin deficiency

Cystic Fibrosis

Friedreich's ataxia

Gaucher's disease

Hypercholesterolemia

Alzheimer's Disease

Autoimmune polyendocrinopathy candidiasis-ectodermal dystrophy

AID—deficiency of activation-induced cytidine deaminase

Ataxia-telangiectasia

CD3-epsilon deficiency (causes SCID)

CD3-gamma deficiency (causes SCID)

chronic granulomatous disease—deficiency of p47^(phox)

Phenylketonuria—Phenylalanine hydroxylase (PAH) deficiency

Tetrahydrobiopterin deficiencies:

GTP cyclohydrolase I (GTPCH) deficiency

6-Pyruvoyl-tetrahydropterin synthase (PTPS) deficiency

Dihydropteridine reductase (DHPR) deficiency

Pterin-4a-carbinolamine dehydratase (PCD) deficiency

Janus Kinase 3 (JAK3) deficiency (causes SCID)

Hereditary fructose intolerance

Porphyria (one of the six forms is caused by a recessive gene)

Sickle Cell Anemia

Tay Sachs syndrome

Thalassemia

Wilson's disease

Xeroderma pigmentosum

Zeta-chain-associated protein kinase deficiency (causes SCID)

The totipotent and/or pluripotent stem cell lines having a homozygousrecessive disease-causing gene that are produced by the methods of thepresent invention are highly useful. They can be cultured underconditions in which they differentiate into cell types related tomanifestation of the disease phenotype. Such cells having a homozygousrecessive disease-causing gene are useful for basic research directed tostudying the disease phenotype ex vivo. They can also be implanted intoexperimental animals (e.g., immunodeficient animals), for study of theirmetabolic activities in vivo. Persons skilled in the art would recognizethat studies in which such cells are genetically modified can be usefulfor gaining understanding of the disease phenotype. Such cells having ahomozygous recessive disease-causing gene can also be used in drugdiscovery; e.g., in screening for drugs or other therapies that willtreat or cure the disease caused by the recessive gene.

In order to further illustrate the invention and its preferredembodiments, the following examples are provided. These examples areintended to be exemplary and in no way limitative of the scope of thepresent invention.

Example 1 Production of Parthogenic Primate Primordial Stem Cells(PPSC's) Materials and Methods

1. Cynomolgus Monkey (Macaca fascicularis) were superovulated using asingle injection of 1000 IU of pregnant mare's serum gonadrophin (PMSG)and 500 IU of human chorionic gonadoprophin (hCG) four days later.

2. Ovaries were retrieved by laparotomy and oocytes dissected from thefollicles and placed in maturation media 36 to 48 hrs after (hCG).Maturation media consisted of medium-199 (Gibco BRL) with Earle'sbalanced salt solution supplemented with 20% fetal bovine serum, 10|U/mlof PMSG, 10 Mimi of hCG, 0.05 mg/ml of penicillin G and 0.075 mg/ml ofsteptomycin sulfate (Hong, 1999).

3. Oocyte Activation

After 40 hrs in maturation, metaphase II eggs were placed in 10micromoles of lonomycin followed incubation in 200 mM 6-DMAP(dimethylaminopurine) for 3 to 4 hrs.

4. Embryo Culture. Commercially available embryo culture media ‘Cooks’was used (modified SOF). Embryos were cultured with a co-culture ofmitotically inactivated mouse embryonic fibroblasts as feeder layer.

5. Isolation of Inner Cell Mass

-   -   a) Upon development to blastocyst, embryos were placed in a        buffered solution of 0.3% pronase for 2 minutes to digest zone        pellucida    -   b) Blastocyts were then rinsed in buffered solution and moved to        solution of 01 culture media and polyclonal antibodies        (antihuman whole serum) 1:3 dilution for 30 minutes.    -   c) Embryos were rinsed 5 times in a buffered solution.    -   d) Embryos were moved into a solution of G1 culture media and        guinea pig complement 1:3 dilution for 30 minutes.    -   e) Remaining of the embryos (dead trophoblast cells and ICM) wee

rinsed 5 times in buffered solution the Inner Cell Mass (ICM) wasisolated and placed on top of a mouse embryonic fibroblast feeder layerfor isolation and growth of Primordial Stem Cells (PSC's).

Results

We have obtained 450 eggs total, after maturation, 224 were still atgerminal vesicle stage (GV=no maturation), 79 were dead, 56 were atmetaphase one (MI) and 91 at metaphase two (Mil).

We have parthenogenically activated all of them. As expected, there wasno cleavage on the GY group, 32% cleavage on the MI and 57% on the MILWhen put in culture, 7 embryos developed to the blastocyst stage (SeeFIG. 1).

After attempting to establish ES-like culture cells, four Inner cellmasses attached nicely one differentiated immediately, and out of thethree remaining, one cell line was obtained. This cell line is calledCyno 1 (FIG. 2). This cell line before and after immunosurgery is shownin FIG. 3 and FIG. 4. FIG. 5 shows the Cyno 1 Cell line five days afterplating.

FIG. 6 shows the Cyno 1 cell line growing on top of a mouse fibroblastfeeder layer. These cells show typical morphology ofpluripotent-embryonic-cells such as small nuclear cytoplasmic ration andthe presence of cytoplasmic granules.

These cells were maintained in an undifferentiated state for a period ofmonths. This is evidenced by screening of such cells after prolongedculturing for the expression of a cell marker characteristic ofundifferentiated cells, Alkaline Phosphatase. As expected, cells werepositive on passage 3 and on passage 5.

The fact that these cells maintain their pluripotency is also shown bytheir spontaneous differentiation into many differentiated cell typesafter being placed in tissue culture in the absence of a feeder layer.In the days following, the cells were observed to differentiate intocuboidal epithelium, fibroblasts, beating myocardial cells and othercells. Two colonies of beating myocardial cells were observed in onewell of a 4-well tissue culture plate.

To determine whether differentiated cells of various somatic celllineages were observed from the differentiating PPSC's, we extractedmRNA from differentiated cell cultures, performed RT-PCR, using humansequence primers specific for various differentiated cell types. Asshown in FIG. 6, transcripts of a predicted size for themesodermally-derived transcripts brachyury and skeletal muscle myosinheavy polypeptide 2 were observed. The transcript sonic hedgehogessential for endoderm development was observed. In addition, theneuron-specific ectoderm marker enolase was observed as well as keratin(not shown) as markers of ectodermally derived cells. These PCR productswere not observed in the mouse feeder layer controls or in the absenceof reverse transcriptase.

To establish that the imprinting status of parthogenetic PPSC'sdifferent than that of di-parental PPSCs we looked at the expression ofseveral imprinted genes. Genes that are mono-allelically expressed fromthe paternal allele, would not be expected to be expressed inparthogenetic cells, as these cells are derived exclusively from thematernal genome. The Snrpn gene is mono-allelically expressed from thepaternal allele in mouse blastocyst inner cell mass [Szabo, P E andMann, JR; Genes & Development 9:3097-3108 (1995)]. We looked at theexpression of this gene in the parthogenetic Macaca facicularis PPSCsand found that the express was undetectable by RT-PCR, whereas underidentical conditions, this gene is readily detected in fibroblast cellcultures from the same species. The Snrpn gene is expected to beexpressed in diparental PPSCs, as these cells contain a paternal allele.In FIG. 7, the expected size RT-PCR product for the Snrpn gene is 260bp.

Example 2 Stable Engraftment of Homozygous Fibroblasts inHistocompatible or Non-Histocompatible Cynomolgus Recipients

Connective tissue fibroblasts are generated from the cyno-1 stem cellline described above which are labeled with green fluorescent protein(GFP) gene. This cell line is homozygous as evidenced by the portion ofa single allete of 225 basepairs using a primer set specific forDQBlu6011-17. These cells are propagated in vitro until several millioncells are obtained.

Thereafter, approximately a million labeled connective fibroblasts aretransplanted into histocompatible cynomolgus monkey recipients, andnon-histocompatible cynomolgus controls. Each monkey is transplantedwith a million labeled cells administered by injection in the upper armat for different sites, in four equal parts.

The degree of engraftment of these engrafted labeled cells is assessedat three different times, at four weeks, six months and a year. Three ofthe four grafts are removed at three different times and the number ofGFP labeled cells is determined in the histocompatible transplantrecipients and controls. The number of GFP cells is compared for bothgroups.

Also, a histological examination is effected to look for any signs oflymphocyte infiltration and any signs of rejection.

Example 3 Production of Homozygous Stem Cell Lines from RabbitParthenogenically Activated Oocytes

Rabbit ES cells were similarly obtained from parthenogenetic embryos.Specifically, rabbit oocytes were obtained from superovulating rabbitsand were actuated using ionomycin and DMAP. This resulted inblastocystes, the inner cell masses of which were transferred tofibroblast factor layer. This in turn resulted in the production ofrabbit ES cell lines which stained positive for characteristic embryonicantigens and which gave rise to various differentiated cell types whenremoved from the front layer.

More specifically, true rabbit ES cell lines morphlogically looked likeES cells and differentiated [into] into all three germ cell lineages.Among the cell types that observed from this cell line were myocordial,vascular endothalial, neuronal, and hemotopoiath cell lineages.

Example 4 Protection of Homozygous Stem Cell Lines from HumanParthenogenically Activated Oocytes Production of Autologous Cells byParthenogenetic Activation of Oocytes

Oocytes from three volunteers were used for parthenogenetic activation.The donors were induced to superovulate by 11 days of low dose (75 IUbid) gonadotropin injections prior to hCG injection. A total of 22oocytes were obtained from the donors 34 hours after HCG stimulation,and were activated at 40-43 h after hCG stimulation.

The oocytes ere activated on day 0, using the ionomycin/DMAP activationprotocol described above. Twelve hours after activation, 20 oocytes(90%) developed one pronucleus and cleaved to the two-cell to four-cellstage on day 2. On day 5 of culture, evident blastocoele cavities wereobserved in six of the parthenotes (30% of the cleaved oocytes) thoughnone of the embryos displayed a clearly discernible inner cell mass. Theresults of parthenogenetic activation of the human oocytes aresummarized in Table 4.

TABLE 4 Parthenogenetic Activation of Human Oocytes Embryos with No. ofPronucleus Cleaved blastocoele Cavity Donor Oocytes (%)^(a) (%)^(a)(%)^(b) 1 5 4 (80) 4 (80) 2 14 13 (93) 13 (93) 4 (31) 6 5 3 3 (100) 2(67) Total 22 20 (90) 20 (90) 6 (30) ^(a)As a percentage of activatedoocytes. ^(b)As percentage of cleaved oocytes.

FIG. 8 shows MI I oocytes at the time of retrieval. FIG. 9 shows four-tosix-cell embryos 48 h after activation. Distinguishable single-nucleatedblastomeres (labeled “n” in FIG. 6) were consistently observed. FIG. 10shows embryos with blastocoele cavities (arrows) that were detected onday 6 and maintained in culture until day 7. The scale bars for FIG. 6,FIG. 7 and FIG. 8=100.

In a study similar to the one described above, human oocytes wereactivated using the ionomycin/DIVIAP activation protocol and werecultured in vitro. One of the activated embryos developed a pronucleus,cleaved, formed a blastocoele cavity, and then developed into ablastocyst having an inner cell mass, shown in FIG. 11. The inner cellmass was isolated and plated on mouse feeder layers as described(Cibelli, J. B., et al. 2002. Parthenogenetic stem calls in non-humanprimates. Science 295: 819). The cultured ICIM cells increased in numberover the first week, and cells indistinguishable from human embryonicstem cells were observed. These grew in close association as a colonywith a distinct boundary, as shown in FIG. 12; they had a highnuclear-to-cytoplasmic ratio, prominent nucleoli, and were observed todifferentiate in vitro into multiple differentiated cell types.

Example 5 Production of Homozygous Stem Cell Lines from MouseParthenogenically Activated Oocytes

Using substantially the same methods described in the presentapplication, another research group, Lin et al., Stem Cells 21:152-161(2003) incorporated by reference in its entirety, generated stem celllines from unfertilized mouse metaphase II oocytes. These oocytes wereactivated by 5 minute exposure to 5 mm calcium ionophore (ionomycin)followed by a 3 hour exposure to 6-methyldiaminopurine (DMAP). Thosestem cell lines were characterized as stem cell lines based on theirexpression of characteristic embryonic antigens (SSEAs, OCT-4, alkalinephosphatase and telomerase) and their pluripotency (give rise toectodermal, endodermal and mesodermal cell types).

Specifically, activated, unfertilized oocytes from F1 hybrid mice(H-2-B/DO were used to establish those stem cell lines homozygous forH-2-B and H-2-D respectively. The stem cell lines appearedkaryotypically normal. When cultured in vitro in the pressures ofspecific growth factors, these cell lines gave rise to ectodermal,mesodermal, and endodermal cell types. Histological examination ofcultures revealed cells having the morphology of neuronal cells andhemotopviette lineages (lymphocytes, monocytes and erythrocytes).

Further, when these cell lines were implanted in the kidney ofsyngenetic F1 mice they similarly resulted in teratomas that comprisedcells of all three germ layers. The teratomas when histologicallyexamined showed evidence of hair follicles, thyroid glands, lungepithelium and connective tissue.

CONCLUSIONS

The results in the foregoing examples provides proof of principle,namely that homozygous stem cell lines may be generated from embryos,e.g., parthenogenically activated embryos, and used to producedifferentiated cell types for cell therapy. More specifically, thepresent instruction provides methods for making libraries or banks ofstem cell lines that are homozygous for specific MHC alleles. Thereby, abank of cells is available which can be used to produce differentiatedcells which are histocompatible for a wide range of transplantrecipients. This is feasible with a relatively few number of stem celllines given that certain HLA haplotyes are expressed with relativelyhigh frequency in the human population.

These differentiated cells should be well tolerated and be stablyengrafted given their antigenic expression relative to the transplantrecipient. Also, in the case of stem cell lines derived fromparthenogenically activated oocytes, these cells eliminate certainethical issues with therapeutic cloning, namely a viable embryo (capableof giving rise to an offspring) is never obtained or destroyed. Thesecells are useful for treating any condition wherein cell or tissuetransplantation is therapeutically desirable, e.g., immune deficiencies,age-related deficiencies, cancer, autoimmune disorder, organdeficiencies, disease, or injury, burn, malignancy, cell proliferationdisorders, hemotopoietic disorders, e.g., blood malignancy such asnon-Hodgkins lymphoma, leukemia, inflammatory disorders, connectivetissue disorder, dermatological disorder, ischemia, stroke, neurologicaldisorders and the like. The present cell banks on particularly wellsuited for treating acute disease, particularly when there is notsufficient time to do therapeutic cloning. For example, those cells areuseful in obtaining differentiated cells for treatment of conditionswhere the patient is near death, e.g., sepsis, stroke and otherconditions where cell therapy is urgently needed. Also, the inventionprovides means for having cells on hand that express desired therapeuticpolypeptides which are histocompatible.

1-62. (canceled)
 63. A method for treatment, preferably acute treatment,comprising transplanting cells or tissue that are homozygous for atleast HLA one allele in a person in need of such a transplant,comprising: a. identifying the MHC alleles of a person in need of atransplant (the recipient); b. obtaining from a stem cell bankcomprising a plurality of stem cells homozygous for at least one MHCallele of the transplant recipient; c. generating cells or tissuesuitable for transplant from said stem cells; and d. transplanting saidcells or tissue suitable for transplant into said recipient.
 64. Themethod of claim 63 wherein said stem cell bank comprises at least 10different human stem cell lines, wherein each of said stem cell linesare homozygous for a different combination of HLA alleles relative tothe other stem cell lines.
 65. The method of claim 63 wherein said stemcell bank comprises at least 15 different human stem cell lines whereineach of said stem cell lines are homozygous for a different combinationof HLA alleles relative to the other human stem cell lines in the cellbank.
 66. The method of claim 63 wherein said stem cell bank comprisesat least about 100 to 1000 stem cell lines each homozygous for adifferent combination of HLA alleles relative to the other human stemcell lines in the cell bank.
 67. The method of claim 63 wherein one ormore of said human stem cell lines are ES or inner cell mass-derivedstem cells.
 68. The method of claim 63 wherein one or more of said humanstem cell lines is derived from a parthenogenetic human embryo.
 69. Themethod of claim 63 wherein one or more of said human stem cell lines areproduced by haploidization comprising the steps of a) inserting orfusing a somatic donor cell or nucleus thereof into or with an oocytewhich is treated to remove or destroy its endogenous genomic DNA before,during or after insertion or fusion; b) activation of the reconstructedembryo to expel haploid genome into a pseudopolar body; c) screening ofthe pseudopolar body for the genetype of the remaining pronuclear; d)combination of the two pronuclei to generate a reconstructured diploidembryo by pronuclear transfer or alternatively producing a diploidembryo by transferal of a pronucleus to an activated haploid oocytecomprising desired haploid genome; e) optionally injecting human morulastage embryo lysates into the reconstructed embryos; and f) isolatinghuman stem cell lines from said reconstructed diploid embryo.
 70. Themethod of claim 63 wherein one or more of said human stem cell lines isproduced by the insertion of first and second polar bodies into arecipient cell.
 71. The method of claim 63 wherein at least one of saidstem cell lines is produced by de-differentiation of a somatic cell bycytoplasmic transfer.
 72. The method of claim 63 wherein said human stemcell bank comprises cells which are homozygous for one of the followingHLA serotypes: HLA-A1, HLA-A3, HLA-A11, HLA-A15, HLA-A22, HLA-A27,HLA-A28, HLA-A29, HLA-A32, HLA-B5, HLA-B7, HLA-B8, HLA-B12, HLA-B17,HLA-B18, HLA-B35 and HLA-B40.
 73. The method of claim 63 wherein saidhuman stem cell bank comprises stem cells which are homozygous for atleast one of the following HLA-A, —B or -DR haplotypes: 1, 7, 2; 1, 8,3; 2, 14, 1; 2, 35, 4; 2, 35, 8; 2, 44, 4; 3, 7, 2; 3, 7, 4; 3, 7, 8; 3,35, 1; 31, 51, 4; and 32, 14,
 7. 74. The method of claim 72 wherein saidcell lines are O-negative.
 75. The method of claim 73 wherein said celllines are O-negative.
 76. The method of claim 63, wherein step bcomprises obtaining stem cells selected from the group consisting oftotipotent, nearly totipotent, and pluripotent stem cells.
 77. Themethod of claim 63, wherein step b comprises obtaining embryonic stemcells.
 78. The method of claim 63, wherein step b comprises obtainingstem cells that can differentiate into hematopoietic stem cells.
 79. Themethod of claim 63, wherein step b comprises obtaining hematopoieticstem cells from the stem cell bank.
 80. The method of claim 63, whereinstep b comprises obtaining stem cells homozygous for an MHC alleleselected from HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, and HLA-DP.
 81. Themethod of claim 63, wherein step b comprises obtaining stem cellshomozygous for the MHC alleles encoding HLA-A, HLA-B, and HLA-DR. 82.The method of claim 63, wherein step b comprises obtaining stem cellsderived from embryos produced by in vitro fertilization orintracytoplasmic sperm injection.
 83. The method of claim 63, whereinstep b comprises obtaining diploid stem cells derived from embryosproduced by parthenogenesis.
 84. The method of claim 83, comprisingobtaining diploid stem cells in which all of the MHC alleles arehomozygous.
 85. The method of claim 63, wherein step b comprisesobtaining stem cells derived from embryos produced by cloning by nucleartransfer.
 86. The method of claim 85, comprising obtaining rejuvenatedstem cells.
 87. The method of claim 86, comprising obtaining rejuvenatedstem cells having telomeres that are on average at least as long as thetelomeres of age-matched control cells of the same type that are notgenerated by nuclear transfer techniques.
 88. The method of claim 86,comprising obtaining rejuvenated stem cells for which the proliferativelife-span is at least as long as the proliferative life-span ofage-matched control cells of the same type that are not generated bynuclear transfer techniques.
 89. The method of claim 86, comprisingobtaining rejuvenated stem cells for which the proliferative life-spanis longer than the proliferative life-span of age-matched control cellsof the same type that are not generated by nuclear transfer techniques.90. The method of claim 86, comprising obtaining rejuvenated stem cellshaving EPC-1 activity that is greater than EPC-1 activity in age-matchedcontrol cells of the same type that are not generated by nucleartransfer techniques.
 91. The method of claim 86, comprising obtainingrejuvenated stem cells having telomerase activity that is greater thantelomerase activity in age-matched control cells of the same type thatare not generated by nuclear transfer techniques.
 92. The method ofclaim 85, comprising obtaining stem cells comprising non-humanmitochondria.
 93. The method of claim 63, wherein step b comprisesobtaining stem cells having DNA that is genetically modified relative tothe DNA of the human donor from which the stem cells are derived. 94.The method of claim 92, comprising obtaining genetically altered stemcells, the DNA of which is modified by adding, modifying, substituting,or deleting one or more DNA sequences.
 95. The method of claim 93,comprising obtaining genetically altered stem cells, the DNA of which ismodified so as to obtain, increase, decrease, inhibit, or otherwisemodify, the expression of a gene that is native to or introduced intosaid cells, relative to expression of said gene in a control cellwithout the genetic modification.
 96. The method of claim 93, comprisingobtaining genetically altered stem cells, the DNA of which is modifiedby homologous recombination.
 97. The method of claim 93, comprisingobtaining genetically altered stem cells, the DNA of which is altered toprevent the expression of a gene encoding an antigenic protein thatelicits an immune response contributing to rejection.
 98. The method ofclaim 96, comprising genetically altered stem cells, the DNA of which ismodified so as to inhibit production of at least one HLA antigen bycells of said cell line.
 99. The method of claim 96, comprisinggenetically altered stem cells, the DNA of which is modified so as toinhibit production of one or more HLA antigens selected from HLA-A,HLA-B, HLA-C, HLA-DR, HLA-DQ, and HLA-DP.
 100. The method of claim 96,comprising genetically altered stem cells, the DNA of which is modifiedso as to inhibit production of β2-microglobulin.
 101. The method ofclaim 96, comprising genetically altered stem cells, the DNA of which isaltered by replacing a non-homozygous MHC allele with one that ishomozygous. 102-133. (canceled)